Novel HCN2 Mutation Contributes to Febrile Seizures by Shifting the Channel’S Kinetics in a Temperature- Dependent Manner

Novel HCN2 Mutation Contributes to Febrile Seizures by Shifting the Channel’s Kinetics in a Temperature- Dependent Manner

Yuki Nakamura1,2, Xiuyu Shi2,3, Tomohiro Numata4, Yasuo Mori4, Ryuji Inoue5, Christoph Lossin6, Tallie Z. Baram7, Shinichi Hirose1,2* 1 Department of Pediatrics, Faculty of Medicine, Fukuoka University, Fukuoka, Japan, 2 The Research Institute for the Molecular Pathomechanisms of Epilepsy, Fukuoka University, Fukuoka, Japan, 3 Department of Pediatrics, Chinese PLA General Hospital, Beijing, China, 4 Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, Japan, 5 Department of Physiology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan, 6 Department of Neurology, School of Medicine University of California Davis, Sacramento, California, United States of America, 7 Departments of Anatomy & Neurobiology, Pediatrics, and Neurology, University of California Irvine, Irvine, California, United States of America

Abstract

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channel-mediated currents, known as Ih, are involved in the control of rhythmic activity in neuronal circuits and in determining neuronal properties including the resting membrane potential. Recent studies have shown that HCN channels play a role in seizure susceptibility and in absence and limbic epilepsy including temporal lobe epilepsy following long febrile seizures (FS). This study focused on the potential contributions of abnormalities in the HCN2 isoform and their role in FS. A novel heterozygous missense mutation in HCN2 exon 1 leading to p.S126L was identified in two unrelated patients with FS. The mutation was inherited from the mother who had suffered from FS in a pedigree. To determine the effect of this substitution we conducted whole-cell patch clamp electrophysiology. We found that mutant channels had elevated sensitivity to temperature. More specifically, they displayed faster kinetics at higher temperature. Kinetic shift by change of temperature sensitivity rather than the shift of voltage dependence led to increased availability of Ih in conditions promoting FS. Responses to cyclic AMP did not differ between wildtype and mutant channels. Thus, mutant HCN2 channels cause significant cAMP-independent enhanced availability of Ih during high temperatures, which may contribute to hyperthermia-induced neuronal hyperexcitability in some individuals with FS.

Citation: Nakamura Y, Shi X, Numata T, Mori Y, Inoue R, et al. (2013) Novel HCN2 Mutation Contributes to Febrile Seizures by Shifting the Channel’s Kinetics in a Temperature-Dependent Manner. PLoS ONE 8(12): e80376. doi:10.1371/journal.pone.0080376 Editor: Bernard Attali, Sackler Medical School, Tel Aviv University, Israel Received April 10, 2013; Accepted October 2, 2013; Published December 4, 2013 Copyright: ß 2013 Nakamura et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by a Grant-in-Aid for Young Scientists (B) (21791048 and 24791096 to Y.N.), Grant-in-Aid for Scientific Research (A) (24249060 to S.H.), Grant-in-Aid for Challenging Exploratory research (25670481 to S.H.), Bilateral Joint Research Projects (S.H.) from Japan Society for the Promotion of Science (JSPS), Grants for Scientific Research on Innovative Areas (221S0002 and 25129708 to S.H.) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), MEXT-supported Program for the Strategic Research Foundation at Private Universities 2013–2017 (S.H.), a Grant-in-aid for the Research on Measures for Intractable Diseases (No. H23-Nanji-Ippan-78 to S.H.) from the Ministry of Health, Labor and Welfare, Intramural Research Grant (24-7)for Neurological and Psychiatric Disorders of NCNP (S.H.), the Joint Usage/Research Program of Medical Research Institute, Tokyo Medical and Dental University (S.H.), grants from The Mitsubishi Foundation (S.H.) and Takeda Scientific Foundation (S.H.), 2013–2017 ‘‘Research grants for Central Research Institute for the Molecular Pathomechanisms of Epilepsy of Fukuoka University’’ (S.H.), Recommended Projects of Fukuoka University (117016 to S.H.), and NIH grants NS35439 and NS078279 (T.Z.B.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected]

Introduction A role for Ih in abnormal neuronal excitability has been documented [14,17,19,21–25]. In addition to thalamocortical Hyperpolarization-activated cyclic nucleotide-gated (HCN) seizures [14,26], HCN channels have been implicated in limbic channels are nonselective cation channels in the heart and brain seizures [25,27–29], including those that arise after prolonged [1–3], and are encoded by four genes (HCN1, 2, 3, and 4) [4]. febrile seizures [27]. In support of a role of HCN2 channels in HCN2 protein is expressed in regions including thalamus, epileptic seizures, a mutation in HCN2 was recently causally linked brainstem, cerebral cortex, hippocampus, and amygdala [5,6]. to general epilepsy [30]. In hippocampus and cortex, HCN2 may co-assemble with HCN1, Febrile seizures (FS) are the most common seizures in young at least in part, to form heterotetrameric channels [7–13], which children, and mutations in ion channels have been found in contributes to native Ih, a hyperpolarization-activated cation variants of FS that include epilepsy such as Dravet syndrome and current. Unlike in the thalamus, where Ih contributes to regulating Genetic epilepsy with FS plus (GEFS+), a clinical subset of FS [31– rhythmic activity [1,14], hippocampal and cortical Ih contributes 34]. However it remains unknown if abnormalities in HCN to the resting membrane potential [1,15,16], and is involved in channel function contribute to typical FS. dendritic summation and other parameters of neuronal excitability The aims of this study were to (1) identify novel HCN2 [17–20]. mutations in children with FS, (2) examine differential tempera-

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Figure 1. Genetic analysis of S126L. A left, Family pedigree; the proband is indicated by an arrow. A right, Double-strand partial sequencing data for HCN2 along with the amino acid translation for the unaffected father (I-1) and the mother and child with febrile seizure (I-2 and II-1). The missense mutation c.377C.T (S126L) is indicated by the arrow. B, Putative transmembrane topology of one HCN2 subunit showing the approximate position of S126 in the N-terminus. C, Sequence alignment of the N-terminus. The rectangle indicates the altered residue. doi:10.1371/journal.pone.0080376.g001 ture sensitivity between wildtype and mutant HCN2, and (3) genesis employed double-overlap PCR. The mutagenic primers examine the putative role of HCN2 mutations in FS. We analyzed were (forward) 59-GAGCTCGGATCCACTAGTC- HCN2 for mutations in 160 children with FS, and identified one CAGTGTGGTGG-39 and (reverse) 59-GCCCCGCGGCACaA- novel mutation in two individuals with FS. Characterization of the GAACGACACCT-39, where the altered base (c.377C.T) HCN2 mutant channels showed that the activation kinetics of encoding the p.Ser126Leu (abbreviated as S126L hereafter) mutant HCN2 were faster in hyperthermic environments, exchange is underlined and shown in lowercase letters. compared with the wildtype, increasing the availability of Ih. Because HCN2-mediated Ih promotes neuronal firing, the results Expression of HCN channels and electrophysiological suggest that, in the presence of mutant HCN2, fever would induce recordings neuronal hyperexcitability promoting FS. HEK293 cells were grown in Eagle’s Minimum Essential Medium (MEM) containing 0.1 mM non-essential amino acids Materials and Methods and 10% fetal bovine serum at 37uC in a humidified 5% CO2 Ethics statement environment. Transient expression of HCN2 was achieved by The present study was approved by the Ethics Review transfection of 1.0 mg plasmid DNA with Lipofectamine2000 Committees of Fukuoka University. Parents of each patient and (Invitrogen); pIRES2-EGFP (Clontech, Mountainview, CA) was the parents themselves provided signed informed consent before co-transfected as a fluorescence marker in a 1:10 mass ratio. In the study. experiments where wildtype (W) and mutant DNA (M) were transfected simultaneously, equal amounts of both were transfect- Sample collection and mutation screening ed totaling 1.0 mg. Assuming random assembly and equal subunit This study was performed in Japan. 160 Japanese children supply, stochastically, this produced a mix of different channel diagnosed with FS based on the guidelines of the American types with the following distribution: MMMM and WWWW: Academy of Pediatrics [35] and 125 healthy control subjects were 6.25% each; MWWW and WMMM in all 4 iterations 25% each, examined. The eight exons and exon/intron boundaries of HCN2 MMWW: 37.5% [36]. Three hours after transfection, the cells were amplified by polymerase chain reaction (PCR). The were dissociated with 0.25% trypsin/EDTA (Invitrogen) and sequences were analyzed using a direct sequencing method with seeded onto coverslips. a 3130xl Genetic Analyzer (Applied Biosystems). Electrophysiological analyses commenced 24–48 hrs after transfection on a fluorescence-enabled inverted microscope HCN2 mutagenesis (Nikon, Japan) equipped with a temperature-controlled TC-324B Human HCN2 cDNA was a gift from Dr. A. Ludwig (Friedrich- recording chamber (Warner Instruments, Holliston, MA). The Alexander-University, Erlangen-Nuremberg, Germany). This cells were continually superfused with extracellular solution construct contains the HCN2 open reading frame described by containing (in mM): 110 NaCl, 30 KCl, 0.5 MgCl2, 1.8 CaCl2, GenBank reference sequence NM_001194.3. Site-directed muta- and 5 HEPES, at pH 7.4 (NaOH). Recording pipettes were pulled

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Figure 2. Current traces and activation curves of HCN2 wildtype, S126L and heretomeric channels at 25, 35 and 386C. A,Whole-cell currents of HCN2 wildtype (top), S126L (middle), and heteromeric (bottom) channels at 25 and 38uC. The currents were recorded in response to voltage pulses from a holding potential of 240 mV to test potentials between 2140 to 240 mV. B, C, D, Conductance-voltage curves for wildtype, S126L and heteromeric channels at 25, 35 and 38uC. Continuous lines show Boltzmann fits of the experimental data. doi:10.1371/journal.pone.0080376.g002 from borosilicate glass on a Narishige PC-10 (Japan), and fire- (,3 mV, [37]) and corrected, along with the pipette capacitance, polished on a Narishige MF-830 microforge. Filled with internal using the amplifier’s circuitry (EPC-8, Heka Instruments, Bell- solution comprising (in mM) 130 KCl, 10 NaCl, 0.5 MgCl2,1 more, NY) prior to recording. Data sampled at 7 kHz were 7-pole EGTA, and 5 HEPES, at pH 7.4 (KOH), the pipettes had Bessel filtered at 3 kHz and digitized with a PCI-6221 digitizer resistances of ,3MV. The liquid junction potential was negligible (National Instruments, Austin, TX). Series resistance was com-

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Electrophysiological characterization at 25 C and effects Table 1. V1/2 for wildtype, S126L and heteromeric channels at u 25uC and 38uC. of temperature on wildtype, S126L and heteromeric HCN2 channels FS, by definition, occur during fever. If S126L causes or 256C386C D change contributes to the phenotype, one would expect that evidence of HCN2 dysfunction presents itself preferentially at or above 38uC. nV1/2 (mV) nV1/2 (mV) To examine the effect of fever on wildtype, wildtype/S126L wildtype 12 295.161.9 8 292.762.0 2.4 (heteromeric) channels, and S126L homomeric channels, we S126L 12 299.062.1 8 290.162.6 8.9* recorded whole cell currents at 25 and 38uC, using 3-s voltage hetero 8 294.961.3 7 291.562.9 3.4 steps to hyperpolarizing potentials (2140 to 240 mV) from a 240 mV holding potential (Fig. 2 A). S126L and heteromeric Data are mean 6 SEM. D =(V1/2 at 38uC)2(V1/2 at 25uC). channels exhibited a voltage-dependent inward current that *indicates p,0.05 compared with the wildtype. resembled wildtype channels at 25 C and 38 C. Cells stimulated doi:10.1371/journal.pone.0080376.t001 u u in this fashion produced voltage-dependent inward currents followed by tail currents when stepped back to the 240 mV hold. pensated by 40–70%. All experiments utilized a holding potential + We used the tail currents (Fig. 2 A, arrows) to deduce the channels’ of 240 mV, which approximated the K Nernst potential voltage dependence and voltage sensitivity of activation (expressed (,238 mV) between 25 and 38uC. Cellular capacitance mea- in Table 1), by normalizing to the maximal amplitude and fitting it surements relied on the amplifier’s readout after cell capacitance to a Boltzmann function (Fig. 2B, C, and D). compensation. Leak subtraction was not necessary because the The half maximal activation voltages (V1/2)at25uC for wildtype holding current at EK+ (240 mV) was commonly #10 pA. (n = 12), S126L (n = 12) and heteromeric channels (n = 8) were 295.161.9, 299.062.1, and 294.961.3 mV, respectively, and Fitting and statistical analysis the respective slope factors (k) were 10.360.9, 10.360.6, and Data were analyzed off-line using the Strathclyde Electrophys- 9.861.9. V1/2 at 38uC for wildtype (n = 8), S126L (n = 8) and iological Software (John Dempster, University of Strathclyde, heteromeric channels (n = 7) were 292.762.0, 290.162.6 and Glasgow, UK, downloadable at http://spider.science.strath.ac. 291.562.9 mV, respectively, and k were 9.961.5, 10.061.0 and uk/sipbs/software_ses.htm). Peak amplitudes from tail currents 8.160.7. acquired immediately after stepping back to the holding potential No significant differences were found when V1/2 of the wildtype, (240 mV) were normalized to the maximal current, plotted S126L and heteromeric channels were compared at same against the test potential, and fitted with a Boltzmann function temperature. However, raising the temperature (from 25uCto 38uC) led to depolarizing shift (DV1/2) in S126L channels (DV1/ 2 = +8.9 mV) that was significantly larger than in wildtype ~ 1 y V {V channels (DV1/2: 2.4 mV, p,0.05), which speaks for increased z 1=2 1 exp k temperature sensitivity in the mutant (Table 1). The current- voltage relationship was determined by the steady-state current in response to step pulse stimuli for 3 seconds (Fig. 3 A, B). At 25uC, where y is the normalized tail current, V is the test potential, V is the 1/2 the current density of S126L at 2140 mV (135.9616.2 pA/pF) half-activation potential and k is the slope factor. Activation kinetics was significant larger than that of wildtype (72.1614.6 pA/pF, were fitted with the double-exponential function y = A ?exp(2t/ fast p,0.05). Although there was no significant difference between the t )+A ?exp(2t/t )+C. Data are presented as mean 6 SEM. fast slow slow channels at 38uC, the current density of S126L at 2140 mV Statistical analysis employed one-way ANOVA, and significance was (150.2627.1 pA/pF) and that of heteromeric channels p, set at 0.05. (135.6630.4 pA/pF) were larger than that of wildtype (93.3618.0 pA/pF, p.0.05) (Fig. 3B). Current traces of these Results channels were fitted by two exponential functions. The fast and HCN2 gene analysis slow time constants (tau fast and tau slow, respectively) describing the activation kinetics at 25uC were voltage-dependent and smaller An HCN2 mutation was identified as a heterozygous missense (faster) at more hyperpolarized voltages (Fig. 3 C, top). Between mutation (c. 377C.T) in two unrelated children with FS. The 290 and 2100 mV, tau fast was significantly larger (slower) in mother (I–2), who also suffered from FS in childhood, presented S126L channels (at 2100 mV: 252.6613 ms, n = 10 wildtype, with the same HCN2 alteration as her child (II–1); the father (I–1), compared with 391.9664.3 ms, n = 10 S126L, p,0.05, Table S1). unaffected, did not, suggesting that the change was inherited (Fig. 1 In 290 mV, tau fast was significantly larger in heteromeric A). The mutation is predicted to substitute a polar serine at channels (295.1629.0 ms, n = 10 wildtype, compared with position 126 with a non-polar leucine (S126L). Position 126 is 473.9672.4 ms, n = 7 hetero, p,0.05) within the intracellular N–terminus, close to the S1 trans- Tau fast and slow at 38uC (Fig. 3 C, bottom) also exhibited membrane segment (Fig. 1B). The amino acid in this position voltage dependence and were faster (smaller) than those observed varies among the four different human HCN channels. However, at 25uC. In contrast to the observation at 25uC, tau fast of S126L an alignment of HCN2 protein sequences across species shows that and heteromeric channels was faster than that of wild type from p.Ser126 is evolutionarily conserved (Fig. 1C). This is reiterated by 290 to 2140 mV. This phenomenon was most prominent at the results of a SIFT in silico analysis, which predicts S126L to be 290 mV. To better characterize this behavior, we subtracted tau damaging to HCN2 function [38]. Mutations in other FS fast at 38uC from that of 25uCin2140, 2100 and 290 mV (Fig. 3 candidate genes such as SCN1A, SCN2A, SCN1B, SCN2B, GABRA1, D). In 2140 mV, ‘‘tau fast (25uC) – tau fast (38uC)’’ (Dtau) of GABRB2, and GABRG2 were not found in any of the patients. wildtype was nearly equal to that of S126L and heteromeric channels. However at 2100 and 290 mV, Dtau of S126L and heteromeric channels was larger than that of wildtype channels.

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Figure 3. Kinetic properties in HCN2 wildtype, S126L and heteromeric channels at 25 and 386C. A, B, Current density at 25 (A) and 38uC (B). A, The current density of S126L channels at 25uC is significantly larger than that of wildtype at 2140 mV (*p,0.05). C, Fast activation time constants for wildtype, S126L and heteromeric channels at 25 and 38uC. { denotes tau fast of S126L channels at 25uC is significantly larger than that of wildtype channels at 2100 mV. * denotes tau fast of S126L channels at 25uC is significantly larger than that of wildtype channels at 290 mV. ** denotes tau fast of S126L channels at 25uC is significantly larger than that of heteromeric channels at 290 mV. {{ denotes tau fast of S126L channels at 38uC is significantly smaller than that of wildtype channels at 290 and 2100 mV ({, *, **, {{ p,0.05). D, Alterations in tau fast by increasing temperature at 2140, 2100 and 290 mV. E, F, Relative amplitudes of fast exponential component as function of voltage at wildtype, S126L and heteromeric channels at 25 (E) and 38uC(F). E, Relative amplitude of S126L channels at 25uC was significantly larger than that of wildtype channels at 290 mV (* p,0.05). F, Relative amplitude of S126L and heteromeric channels at 38uC was significantly larger than that of wildtype channels at 290 mV (*, **p,0.05). doi:10.1371/journal.pone.0080376.g003

These results indicate that the shift of the activation kinetics for contribution of the fast component to total Ih in each channel at S126L and heteromeric channels due to raising the temperature is 38uC was less voltage dependent than at 25uC (Fig. 3 F vs. 3 E). larger in mutant channels, and that this is more evident in The contribution of the fast component of S126L and heteromeric relatively depolarized (physiological) voltage ranges. channels was significantly increased compared with wildtype Because the relative contribution of tau fast and slow to the channels at 290 mV (Fig. 3 F, p,0.05). These results suggest that activation kinetics influence channel behavior [7,39], we calculat- the fast component (Afast) in S126L and heteromeric channels is ed the relative amplitude of the fast and slow exponential increased at more physiological potentials and at hyperthermic components of Ih (Fig. 3 E, F, Table S1). At 25uC, in S126L temperatures, and that, compared to wildtype, S126L and channels, the contribution of the fast component was significantly heteromeric channels exhibit altered temperature sensitivity. In higher at 290 mV compared with wildtype channels (Fig. 3 E, summary, the shift of the activation kinetics by a raise in p,0.05). These data indicate that the fast component (Afast)in temperature was highly influenced rather than that of the S126L channels is increased at depolarized voltages. The conductance and the voltage dependence.

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K1/2 and h values for S126L channels were close to these values for wildtype channels (Table S5). These results suggest that the altered profile of S126L channels is a result of changes in voltage dependence and temperature sensitivity rather than in cAMP dependence.

Discussion Here we identified a novel heterozygous HCN2 missense mutation (S126L) in children with FS and characterized its functional properties. We found increased current densities and accelerated kinetics in HCN2 channel with the S126L mutant. These alterations produce a mutant that allows more positive charge to enter the cell more quickly compared to healthy cells, Figure 4. Temperature dependence of HCN2 wildtype, S126L specifically at elevated temperature, resulting in depolarization and heteromeric channels. The effect of temperature on wildtype and excitation. Our results suggest that current mediated by (n = 7–11), S126L (n = 7–12) and heteromeric (n = 7–10) channels at S126L-containing channels may augment neuronal excitability 2140 mV. Elevated temperature raises the current densities in all during hyperthermia. channels, but only S126L-containing channels show a distinctly steeper slope from 35 to 38uC. One may ask whether our findings are truly representative of doi:10.1371/journal.pone.0080376.g004 what is seen in the patients when our data show the strongest effects in setups using only S126L channels, when the patients are Comparison of temperature dependence in wildtype, heterozygous for the alteration. To emulate the patient internal environment, we used setups with a mix of wildtype and mutant S126L and heteromeric channels DNA. This commonly produced behaviors that lay in between To evaluate the temperature dependence in each channel, we wildtype and purely-mutant, which may relate to the stochastic measured the Ih amplitude at 25, 35 and 38uC. Figure 4 shows distribution of the ensuing channel possibilities. Closer analysis of that the current density was increased by a rise in temperature in possible subunit compositions for 1:1 transfection experiments all channels. Between 25 to 35uC, increased current density was reveals that only 37.5% of the channels were composed of 2 similar in S126L (slope: 1.263.0 pA?pF21?K21), heteromeric 21 21 21 21 wildtype and 2 mutant subunits. Exactly half of the channels (50%) (2.063.1 pA?pF ?K ) and wildtype (0.863.0 pA?pF ?K ) contained 3 subunits of one type and the remaining subunit was of channels (Table S2). However, raising the temperature from 35 the other type. Most importantly, however, even in WT/mutant to 38uC - a temperature commonly associated with fever - led to transfections, 12.5% of the channels were in fact homomeric, highly divergent effects: whereas a gradual increase continued in 21 21 either wildtype or mutant. A ‘‘dose-dependent’’ effect is therefore the current density of wildtype (0.6610.1 pA?pF ?K ) channel, to be expected, and our data confirm this, because all heteromeric a drastic, steep increase in current density occurred in the S126L channels produced electrophysiological deviations similar (usually (4.7612.6 pA?pF21?K21) and heteromeric 21 21 somewhat reduced) or equal to what was seen in the setups using (6.7614.7 pA?pF ?K ) channels. These results suggest that only mutant DNA. We have no way of examining the specific modulation by this mutation was occurred between 35 and stoichiometry of the mutant channels in the native environment. 38uC. It is uncertain, for example, whether, in the patients, S126L protein assembles with wildtype and/or mutant protein as readily Role of cAMP in mutant HCN2 channel function as it does with healthy protein. Conversely, we cannot exclude that Because cAMP is a direct modulator of HCN channels [3,40] S126L does not enhance subunit interaction between the mutants, and may influence the availability of Ih during hyperthermia, we which could explain the augmented current densities as we investigated the effects of cAMP on S126L in comparison to elaborate below. wildtype channels. As expected, the activation curves of both In most cortical and hippocampal neurons, Ih channels are channels shifted towards more depolarized potentials following constitutively open near the resting membrane potential [41–43], application of cAMP at 25uC (Fig. 5 A, B). In addition, there was a and contribute to its maintenance [1,16]. In addition, Ih controls tendency for higher sensitivity of the mutant channels to low doses the membrane potential via a depolarizing inward current and by (2 mM) of cAMP (Table S3, p,0.05). facilitating hyperpolarization [44,45]. The large shift of the More specifically, focused on 2 mM cAMP – the lowest dose kinetics in homomeric S126L and heteromeric channels near the leading to consistent changes in the activation curves in all resting membrane potential at high temperatures could lead to channels at 38uC – resulted in a +3.5 mV(p = 0.40) right-shift of early activation of Ih and disrupt this Ih-based fine-tuning, the activation curve for S126L channels. Wildtype channel, on the promoting neuronal hyperexcitability. other hand, showed almost no shift (20.1 mV, p = 0.98, Table S4). In hippocampal CA1 neurons of immature rats, hyperthermia Although no significant difference was noted between the wildtype may reduce GABAA-receptor-mediated synaptic inhibition [46]. and S126L channels, this change produces clear conductance Such decrease, coupled with the shift in the activation kinetics of differences that enables the mutant to mediate more current (Fig. 5 HCN2 S126L and heteromeric channels during hyperthermia C, D). could lead to an imbalance between neuronal excitation and The data produced in Figure 5A and B were re-plotted to inhibition, accelerating the development of FS. produce cAMP dose-response curves based on the half-maximal Increased current densities at strongly hyperpolarized potentials activation (Fig. 5E, F). Hill fits of the data show that S126L have been reported before [24,47,48]. At this time, we cannot channels were activated at more positive voltages than wildtype explain this phenomenon. Three factors contribute to macroscopic channels at 38uC and that both channels at 38uC were activated current (I), namely the number of contributing channels (N), single- by lower cAMP concentrations compared with 25uC. However channel current (i), and open probability (p). We deem it unlikely

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Figure 5. Effects of cAMP concentrations and raising temperature. A, B, Conductance-voltage curves of wildtype (A) and S126L (B) channels in the absence and presence of cAMP at concentrations of 1, 2, and 10 mM. C, D, Comparison of conductance-voltage curves between at 25uC and at 38uCin2mM cAMP of wildtype (C) and S126L (D) channels. E, F, Hill fits of the cAMP dose-response as measured by half-maximal activation at 25uC and 38uC. doi:10.1371/journal.pone.0080376.g005 that either i or p are altered, simply based on the location of known importance of the N-terminus in inter-subunit interaction p.Ser126 within the HCN2 structure. We expect to find p.Ser126 and formation of functional homo- and heteromeric channels [9]. in the periphery of the channel, although a definitive crystal The substitution of a polar serine with a nonpolar leucine might structure is so far unavailable. This position would situate modify intermolecular forces or hydrogen bonds with other amino p.Ser126 far from the pore, which makes it hard to conceive an acids, leading to aberrant interaction with other HCN subunits influence on the HCN2 single-channel conductance, which defines required for tetramerization. Indeed, heteromerization of HCN2 i in a voltage-dependent fashion. Similarly, p.Ser126 would be with HCN1 [9,10] is highly regulated in the brain [12,13]. unlikely to impact the open probability of the channel. Unfortu- Augmented heteromerization after experimental FS [13] has been nately, enhanced surface expression is not a plausible explanation correlated with a shift in Ih properties that is similar to the one we for the increase in Ih either, as this would imply a voltage- found in S126L channels [24]. These data raise the possibility that dependent change in the number of channels in the membrane. S126L enhances HCN2:HCN1 interaction, leading to aberrant There is, however, the possibility of a change in functional-channel channel function. availability. How this could be mediated is currently unclear. The mutation might interfere also with the interaction of HCN2 Possible scenarios, given the residue’s mapping to the N-terminus, with auxiliary proteins. HCN channels interact with filamin [49], include abnormal phosphorylation as well as alterations in protein- TRIP8b [50,51] and enzymes such as p38 MAPK [52] and Src protein interactions. The latter is particular interesting, owing to a kinase [53]. Whereas the large majority of these proteins interact

PLOS ONE | www.plosone.org 7 December 2013 | Volume 8 | Issue 12 | e80376 HCN2 Mutation and Temperature Sensitivity with HCN1 [54], tamalin, KCNE2, and others interact with Table S3 cAMP sensitivity in wildtype and mutant HCN2. S126L might impair these interactions, enhancing a channels: comparison of the shift in the voltage depen- kinetic shift of the channel and promoting FS. dence of activation. The DV1/2 describes the cAMP-induced In conclusion, a mutation in HCN2 has been identified, with a voltage shift in half-maximal activation compared to control levels. potential to contribute to FS in a subgroup of children. It is now * indicates p,0.05 compared to the control. the 3rd HCN mutation found in seizures and epilepsy, all of which (DOC) affect HCN2. This underlies the importance of HCN channels to Table S4 Temperature dependence of cAMP sensitivity normal and aberrant neuronal excitability, and the specific in wildtype and mutant channels. properties of HCN2 that make mutations in these channels (or (DOC) their absence) likely to result in a hyperexcitable network. Table S5 cAMP dose-response curves based on half- Supporting Information maximal activation voltages. (DOC) Table S1 Kinetic parameters for HCN2 current activation at 290 mV based on double-exponential fits. For the Acknowledgments sake of clarity, only fast kinetic parameters are listed. * indicates p,0.05 versus wildtype. We thank Dr. A. Ludwig for providing human HCN2 cDNA. We are grateful to Ms. M. Yonetani and A. Hamachi for their technical assistance. (DOC) Table S2 Development of current density versus tem- Author Contributions perature. The difference in the averaged current densities Conceived and designed the experiments: YN RI TZB SH. Performed the between two temperature points at 2140 mV was calculated as a experiments: YN XS TN. Analyzed the data: YN TN CL. Contributed slope value. reagents/materials/analysis tools: YN YM RI SH. Wrote the paper: YN (DOC) CL TZB SH.

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